1. Field of the Invention
The present invention relates to active transfer of haptens, proteins, peptides, nucleic acids and other molecules into cells. More particularly, the present invention relates to novel polypeptides which can effectively penetrate into cells, in particular eukaryotic cells, and transport thereto a substance of interest which is capable of constituting novel antiviral compositions. This invention is of major importance as it has application in a variety of fields, in particular that of gene therapy and vaccines.
2. Description of the Background
Gene therapy remains dependent on a considerable number of parameters, among them the development of vectors which are capable of transferring active principles endowed with predetermined specific properties to the cytoplasm of cells of the host organism under consideration in the absence of genetic alterations associated with the use of such vectors, and with no degradation of the biological activity of the transferred active principles. Current knowledge is that in spite of the effort achieved in developing vectors of viral or non viral origins, not all of these conditions have been satisfactorily fulfilled.
Further, the possibility of transporting substances efficiently into cells is also important for all biotechnological applications. Thus transferring substances into cells in vitro or ex vivo can be used either to produce proteins or peptides, or to regulate gene expression, or to analyse the properties of a given substance in that cell. In vivo, the transfer of a substance to a cell can also act to create models for studying diseases in animals or for studying the effect of a given compound on an organism.
The present invention thus aims to provide a novel type of vector which is both effective and is more innocuous than viral vectors in current use.
International patent application WO 97/02840 describes the use of antibodies or their F(abxe2x80x2)2 and Fabxe2x80x2 fragments which can penetrate into the interior of living cells, as immunovectors for intracytoplasmic and intranuclear transfer of biologically active substances. While such vectors are highly effective, their use can produce problems in some applications. The use of antibodies or F(abxe2x80x2)2 antibody fragments involves the production of high titers of these molecules with qualities which are compatible with therapeutic use. Further, the use of molecules with the size and complexity of antibodies can constitute a further disadvantage, in particular as regards use. U.S. Pat. No. 5,635,383 illustrates a further type of complex vector based on polylysine for transferring nucleic acids into cells.
The present application relates to novel polypeptides with advantageous properties both for transferring of substances into cells and as antiviral agents. The primary structure of these polypeptides is much simpler than antibodies and they are of reduced size. Further, preparation is easy and their potential applications are highly varied.
More particularly, the present invention stems from the discovery by the inventors that it is possible to identify, from whole antibodies, limited regions carrying a cellular penetration activity. The invention also stems from the discovery that it is possible to isolate, from whole antibodies, in particular from a single chain of these antibodies, peptides or polypeptides endowed with cell penetration activity. The present invention constitutes the first demonstration that a fragment of a single chain of an antibody can effectively penetrate into cells. The present invention also constitutes the first demonstration that such a fragment is also capable, advantageously, of transporting a substance of interest into said cell, and can preferably have an antiviral activity.
The present invention thus provides novel molecules which are particularly adapted to transfer biologically active substances into eukaryotic cells, particularly mammalian cells.
In a first aspect, the invention provides a polypeptide characterized in that:
it is constituted by a unique or repeated peptide motif; and
it comprises an amino acid sequence endowing it with the capacity to penetrate into cells and, if necessary, to transport thereto a substance of interest.
In this regard, the invention concerns a polypeptide characterized in that:
it is constituted by a unique or repeated peptide motif; and
it comprises an amino acid sequence constitute by one or more different antibody fragment(s); and
it is capable of penetrating into cells.
In one implementation of the invention, the polypeptides thus comprise one or more fragment(s) of an antibody which may or may not be different. In their simplest form, antibodies (molecules from the immunoglobulin superfamily) are constituted by four chains which are associated together (for example IgG) two heavy chains H, and two light chains L (FIG. 1). These four chains are associated together post-synthesis to form a molecule with a molecular weight of about 150,000 kD. The antigenic specificity of antibodies is provided by variable domains involving a number of regions of a heavy chain and a number of regions of a light chain (FIG. 1).
Polypeptides can also be constituted by sequences originating from other immunoglobulin representatives such as IgM.
Each heavy chain of an antibody is composed of about 450 amino acids, and comprises different domains termed the constant domain (C), variable domain (V) and joining domains (D and J). Particular motifs are found in the variable domains, termed CDR (Complementarity Determining Region) which can readily be localised by sequence alignment (C. Janeway and P. Travers, 1996, Immunobiology, Academic Press, xe2x80x9cThe Structure of a Typical Antibody Moleculexe2x80x9d). For an analysis of the sequences of the variable regions, reference should also be made to the article by T. T. Wu and E. Kabat (J. Exp. Med., 1970, Vol. 132, p. 211-250). CDR motifs themselves comprise hypervariable regions.
The present application stems from the demonstration that it is possible to obtain regions which are limited in size and of simple structure with particularly advantageous properties from the antibody structure. Thus, starting from a molecule which is complex (four associated chains) and large (150000 kD), the Applicant has succeeded in constructing polypeptides with a single chain, with the capability of penetrating into cells and of transporting thereto substances of interest. The properties of the polypeptides of the invention are all the more remarkable since their sequences corresponding to those of one or more fragments of only one of the chains of an antibody and thus in order to be active, there is no need for constant regions originating from a heavy chain and a light chain. Polypeptides of the invention obtained by chemical synthesis have the same properties.
The term xe2x80x9cpolypeptidexe2x80x9d as used in the present invention defines a molecule comprising a concatenation of amino acids, with a size in the range 3 to 100 amino acids, for example less than 60 amino acids. Still more preferably, it is a molecule comprising a concatenation of 3 to 60 amino acids, advantageously 3 to 30. Particularly preferred polypeptides advantageously comprise more than about 10 amino acids. The polypeptide of the invention can also comprise certain structural modifications, of a chemical or enzymatic nature for example. Thus the polypeptide of the invention can comprise certain functional groups which, by chemical or enzymatic reaction, can couple with another substance. The polypeptides of the invention can also be chemically modified in order to render them more resistant to proteases or less visible to the immune system. The polypeptides of the invention can be obtained by any method which is known to the skilled person, in particular by chemical synthesis, for example using peptide synthesisers, or by fragmentation or deletion from larger polypeptides, natural or otherwise. They can also be prepared using recombinant DNA techniques, by expression of a corresponding nucleic acid in a eukaryotic or prokaryotic host cell. Clearly, they can result from combinations of these different methods.
To this end, the polypeptides of the invention can be produced from libraries of nucleic acids or peptides, such as synthesised combinatorial libraries.
The term xe2x80x9cunique peptide motifxe2x80x9d means that, in contrast to antibodies or Fab or F(abxe2x80x2)2 type antibody fragments, for example, the polypeptides of the invention comprise only a single chain of amino acids. The term xe2x80x9crepeated peptide motifxe2x80x9d means that the polypeptides of the invention can comprise different peptide blocks assembled together, optionally chemically, to form a single chain.
The term xe2x80x9cpenetratexe2x80x9d or xe2x80x9cpenetratingxe2x80x9d as used in the present invention means a polypeptide which is capable of passing from the external medium to the intracellular medium, in particular into the cell cytoplasm. This capacity can be determined in different manners, in particular using a cell penetration test comprising initial incubation of the polypeptide to be studied in the presence of culture cells followed, after fixing and permeabilisation of these cells, by revealing the presence of said polypeptide inside said cell. Revealing can be achieved by a further incubation with labelled antibodies directed against said polypeptide and detection, in the cytoplasm or in the immediate proximity of the nucleus or even in the nucleus, of the antigen-antibody type immunological reaction between the polypeptlde and the labelled antibody. A previously labelled polypeptide of the invention followed by detection of said labelling in these cellular compartments can also be used for revealing. Such a cell penetration test has been described, for example, in International patent application WO 97/02840.
As indicated above, the present invention stems from demonstrating the existence of reduced regions of an antibody endowed with cell penetration properties and which can also act to transport substances of interest. More particularly, the inventors have sought the presence of regions endowed with cell penetration properties and which could be used as a vector in place of whole antibodies in the structure of certain penetrating antibodies such as those described in WO 97/02840. To this end, the inventors have first determined the complete sequence of heavy and light chains of three particular monoclonal antibodies, J20.8, F4.1 and F14.6. These antibodies are anti-DNA antibodies, polyreactive, which are produced by hybridoma deposited at the CNCM [National Collection of Micro-organism Cultures] under numbers I-1605, I-6506 and I-1607 (see patent application cited above). Alignment of these sequences and their comparative analysis have revealed the following remarkable elements:
the existence of a region of very high homology (65-70%) in the CDR2 region of these three antibodies; and
the presence, in these three antibodies, of CDR3 regions which are rich in lysine and arginine (basic amino acids).
With regard to these results, and given that the majority of peptides capable of transport and nuclear localisation are rich in lysine and arginine, the Applicants then synthesised series of polypeptides corresponding to different regions of these antibodies, and in particular to the CDR2 and CDR3 regions, and hybrid constructions in which certain of these regions were fused together (in particular a CDR2-3 peptide carrying CDR2 and CDR3 regions in succession). A biotin residue was also introduced to the N-terminal side of these polypeptides, to enable them to be detected easily.
These polypeptides were then tested for their capacity to penetrate into cells. The results obtained show that, remarkably, certain of these polypeptides have the capacity to penetrate effectively into cells. In particular, the results obtained show that the group of polypeptides which comprise all or a portion of the CDR3 region are capable of penetrating into cells.
More preferably, the polypeptides of the invention are thus constituted by a unique chain comprising at least one fragment of the heavy chain of an antibody. Still more preferably, they comprise at least a fragment of the variable region of the heavy chain of an antibody.
In a particular implementation, the invention concerns polypeptides as defined above comprising all or a portion of the CDR3 region of an antibody.
Further, the results obtained have also shown that polypeptides also containing all or a portion of the CDR2 region also have the capability of penetrating into cells. To this end, polypeptides which combine all or a portion of the CDR3 region and all or a portion of the CDR2 region have entirely remarkable cell penetration capacities.
Thus in a further implementation, the polypeptides of the invention comprise all or a portion of the CDR2 region of an antibody.
In a particularly interesting implementation, the polypeptides of the invention more preferably comprise all or a portion of the CDR3 region and all or a portion of the CDR2 region. This type of polypeptide is particularly advantageous as it is capable of mass penetration into the interior of living cells.
More particularly, the expression xe2x80x9call or a portionxe2x80x9d as used in the present application means that the polypeptides of the invention can comprise either the whole of the CDR region concerned of an antibody, or only a portion thereof, it being understood that the polypeptide retains a cell penetration capacity (functional homologue). A portion of the CDR region can consist of a CDR region which is free of one or more terminal amino acids, in particular one, two or three terminal amino acids. It may also be a CDR region where one or more internal residues have been deleted or substituted by other amino acids, preferably amino acids of the same nature (for example basic amino acids). Advantageously, less than 30% of the internal residues of the CDR region are modified, preferably less than 20% and more preferably less than 15%.
Preferred polypeptides of the invention are thus polypeptides comprising all or a portion of a CDR3 region of an antibody. By way of illustration, CDR3 regions with sequence SEQ ID NO 1, 2, 3, 8 or the sequences shown in FIG. 2 and FIG. 3 or any functional homologue can be cited.
The antibody fragments can themselves constitute the polypeptide of the invention. They can also be modified by adding residues to one or both of their extremities. In particular, it may be advantageous to add amino acids which give the fragment, in particular the CDR region, a better spatial configuration. It may also be advantageous to add one or more essentially basic amino acids, lysine and/or arginine in type, to stabilise the polypeptide and increase its interaction with the cell membranes. Further, as indicated above, the polypeptides of the invention may comprise several regions of an antibody chain, such as a CDR2 region and a CDR3 region. These regions can in particular be fused together or spaced by amino acids as described above.
Particular polypeptides of the invention are polypeptides comprising a CDR3 region of an antibody or polypeptides essentially comprising a fusion between the CDR3 region of an antibody and the CDR2 region of an antibody. Examples of such polypeptides are the CDR3 polypeptides and the CDR2-3 polypeptide the sequences for which are given in the Examples.
Experiments carried out with these polypeptides, in particular polypeptides comprising the CDR3 region, and more particularly those comprising the CDR3 region and the CDR2 region, clearly show that:
1) incubating PtK2, HeLa or 3T3 cells for one hour with complete culture medium (10% foetal calf serum) containing the polypeptide is sufficient for the polypeptide to be massively transported into the cytoplasm of all the cells and into the nucleus of a large proportion of these cells, the proportion being variable depending on the line concerned.
2) When cells are incubated for 2 hours in complete culture medium containing pre-formed peptide-streptavidin complexes coupled to peroxidase (MWxe2x89xa7100000) or peptide-streptavidin coupled to alkaline phosphatase (MWxe2x89xa7180000), the corresponding enzymes are detected in the cytoplasm of all of the cells of the culture and weakly to intensely detected in the majority of the nuclei of these cells. No intracellular coloration is observed when the cells are incubated in the presence of streptavidin coupled with peroxidase, streptavidin coupled with alkaline phosphatase or with streptavidin or the enzymes in their native forms.
The polypeptides of the invention, in particular of type CDR3 and CDR2-3, and their peptide-streptavidin-enzyme complexes are transported in large quantities into a large proportion of human peripheral cells and particularly into activated T lymphocytes.
In general, the polypeptides of the invention can be constructed using different techniques which are known to the skilled person (supra), starting from any given antibody, in particular any given monoclonal antibody.
Preferably, the polypeptides of the invention are obtained by chemical synthesis or are constructed from a fragment or several fragments of one or more penetrating antibody(ies), preferably a penetrating monoclonal antibody. The existence of antibodies which can penetrate inside cells and in particular into the nuclei of human lymphocytes when these cells are incubated in vitro in a culture medium containing a serum originating from patients with disseminated lupus erythematosus (DLE) was reported for the first time by Alarcon-Segovia et al. In 1978 (Nature, 271). Recently, this type of antibody has been detected in the lupus mouse MRL lpr/lpr, but also in the NZB mouse with an autoimmune hemolytic disease syndrome and even in the normal BALB/c mouse. Certain monoclonal antibodies prepared from the spleen of these mice have been shown to be capable of penetrating in vitro into the nucleus of cells maintained in culture (Vlahakos et al., J. Am. Soc. Nephrol. 2 (1992) 1345; Eyal Raz et al., Eur. J. Immuno. 23 (1993) 383). Further, it has been shown that these antibodies are also capable, when injected into mice, of penetrating into several types of cells, and are found in their nuclei (Okudaira et al., Arthritis and Rheumatism, 30 (1987) 669).
In general, any antibody can be selected with a view of determining its penetrating character. This selection can be made, for example, using a cell penetration test comprising initial incubation of the antibody under study in the presence of cells, in particular cells into which it is desired to transport a substance of interest, followed by fixing and permeabilisation of these cells, revealing the presence or the absence of this antibody in the plasmic membrane, the cytoplasm or in the immediate proximity of the nucleus or even in the nucleus. Revealing can, for example, be effected by incubation with a second labelled antibody, directed against the test antibody, followed by detection of the immunological reaction of the antigen-antibody type between these two antibodies. Such a test has been described in detail, for example in French patent FR-9508316.
Still more preferably, the fragment of antibody used to construct a polypeptide of the invention is a fragment of a polyreactive antibody, in particular penetrating and polyreactive. A polyreactive antibody is an antibody which is capable of recognising several different antigens. In general, such antibodies have a particularly high affinity for a particular type of antigen and are capable of recognising one or more other antigens with a lower affinity. The polyreactivity of antibodies can be demonstrated by any conventional immunological technique, such as the methodology described by Sibille et al. (Eur. J. Immuno. 1997, 27: 1221-1228).
Advantageously, the polyreactive antibodies used in constructing the polypeptides of the invention are capable of reacting with nucleic acids, free or complexed with proteins (anti-DNA antibodies). This property can be demonstrated using the ELISA technique or by passing the antibodies over a column or any other support on which DNA has already been immobilised. The anti-DNA antibodies are thus retained on the support and can be eluted and isolated using conventional techniques. In general, the avidity for DNA of the anti-DNA antibodies used in the context of the invention is of the order of 1xc3x97106 M to 2xc3x97107 M. Preferably, these antibodies recognise genomic DNA, in particular genomic DNA. In a particular implementation, the antibodies used are polyreactive antibodies which recognise the genomic DNA of human hematopoietic cells. Still more preferably, they are antibodies which are capable of reacting with nucleic acids and recognise, inter alia, proteins such as Tat from the HIV retrovirus, and/or constituents of the cell surface and of the cell cytoskeleton.
To construct a polypeptide of the invention, the selected antibody is then used as follows:
a) if the sequence of the variable region of the heavy or light chain of this antibody is not accessible, it is determined in a first stage. Conventional sequencing techniques can be used, as illustrated in the examples;
b) the CDR regions are localised by sequence alignment with other sequences of antibody chains, or by any other technique;
c) fragments of this sequence are prepared, or the polypeptides corresponding to regions of this sequence are synthesised, and assembled if necessary. To this end, any conventional technique which is known to the skilled person can be used (peptide sequencers, using a restriction enzyme, ligases, etc . . . );
d) the fragment obtained may be modified by addition, deletion or substitution of amino acids;
e) the cell penetration capacity of the polypeptide obtained is then tested under the conditions described above, also the capacity of transporting substances such as fluorescein or peroxidase.
Optionally, steps c), d) and e) or d) and e) are repeated so as to improve the penetration efficacy or the general properties of the polypeptides of the invention.
In a supplemental subsequent step f), the polypeptide obtained, with the capacity to penetrate into cells, is then used in a coupling reaction with a given substance to generate a vector as will be defined below.
An alternative to this method lies in the use of one or more libraries of nucleic acids or peptides as the starting material. Thus rather than starting from the sequence for an antibody, it is possible to construct, for example by combinatorial chemistry, libraries of peptides or nucleic acids coding for peptides representing functional homologues of the CDR2 or CDR3 antibody regions.
The peptides or combinations of peptides or nucleic acids of these libraries are then prepared, optionally modified and tested for their activity using steps c) to e) of the above method.
Preferably in step c) of the above method, the prepared fragments comprise all or a portion of the CDR3 region of an antibody.
In step d), modifications can, for example, consist of introducing certain supplemental amino acids, either simply for technical reasons (ease of synthesis, coupling between different regions, etc) or for structural or physicochemical reasons. Concerning amino acids for xe2x80x9cfillingxe2x80x9d, amino acids which are relatively neutral on the structural and physicochemical level are advantageously used. Regarding structural reasons, as indicated above, adding residues can improve the conformation of the polypeptide and thus potentialise its activity. As an example, introducing nuclear localisation factor sequences (NLF) can increase the intranuclear transfer potentials. Further, it may also be desirable to increase the basic nature of the polypeptides.
To this end, to improve the compaction properties of the polypeptides of the invention, in particular as regards nucleic acids, polypeptides have been constructed which carry lysine residues on the N-terminal side. Advantageously, the number of lysine residues is less than 30, more preferably between 10 and 20.
The results presented in the examples confirm the penetration properties of these polypeptides, and their capacity for effective transport of substances of interest, in particular nucleic acids.
Whatever the additions made, the polypeptide of the invention as prepared, for example, using the above protocol, preferably comprises at most 100 amino acids. Still more preferably, it comprises 3 to 60 amino acids, preferably 3 to 40 amino acids.
In a further aspect, the invention concerns the use of a polypeptide as defined above to transfer substances into cells, in vitro, ex vivo or in vivo.
In a still further aspect, the invention concerns a vector for transferring a substance into a cell, characterized in that it comprises a polypeptide as defined above to which said substance is coupled.
In one implementation of the invention, the polypeptide comprises a sequence of amino acids endowing it with the ability to penetrate into cells enabling it to transport into said cell substances of biological interest which are associated therewith, for example haptens or macro-molecules of hundreds to thousands of kD, such as drugs, proteins or nucleic acids.
The sequence of amino acids and the substances of therapeutic interest associated with it can, for example, be coupled or bonded via covalent or non covalent bonds.
A polypeptide of the invention is advantageously constituted by peptides or macro-molecules with the capability of penetrating into living cells, and more particularly from peptide derivatives of antibodies or antibody fragments as described in International patent application WO 97/02840 or from other peptides comprising one or more hypervariable antibody portions, or synthetic molecules, not directly related to an antibody type structure, which can be obtained, for example, by screening a peptide library for cell penetration.
A particular polypeptide of the invention is thus composed of a unique or repeated peptide motif, and comprises a sequence of amino acids which endow it with the capacity to penetrate into cells and transport a substance of interest thereto, this sequence being capable of being obtained by screening a peptide library for cell penetration. The conditions for screening such libraries have been described above.
A further particular polypeptide of the invention is composed of a unique or repeated peptide motif and comprises a sequence of amino acids which endow it with the ability to penetrate into cells and transport thereto a substance of interest, this sequence being composed of a peptide comprising one or more hypervariable antibody portions.
The coupled substance can be any product of interest, in particular a biological, pharmaceutical or agro-alimentary product. In particular, it may be a nucleic acid, such as a ribonucleic acid or a deoxyribonucleic acid. This nucleic acid can also be from a variety of origins, in particular human, viral, animal, eukaryotic or prokaryotic, plant, synthetic, etc . . . This nucleic acid can also be a variety of sizes, from a simple oligonucleotide to a genome or a fraction thereof. In particular, it may be a viral genome or a plasmid. The substance can also be a protein, such as an enzyme, hormone, cytokine, apolipoprotein, growth factor, etc . . . A particular type of substance is represented by antigens. As indicated below, the polypeptides of the invention can advantageously act as an adjuvant and stimulate the immune response directed against an antigen.
More generally, the substance can be any active principle of a drug, be it a chemical, biochemical or synthetic product.
To enable its transfer into a cell, said substance is thus coupled to a polypeptide of the invention.
The term xe2x80x9ccoupledxe2x80x9d as used in the invention means any type of interaction enabling a physical association between the substance and the polypeptide. Preferably, however, the interaction is sufficiently stable for the vector not to dissociate before cell penetration. For this reason, the preferred coupling is covalent coupling.
Covalent coupling can be effected by different techniques which are known to the skilled person. In particular, it can be effected using maleimide, succinimide, peptide, disulphide and thioether bonds. Reference should be made in this respect to xe2x80x9cBioconjugate Techniquesxe2x80x9d by Greg T. HERMANSON (Academic Press, 1996).
A particular method consists, for example, of adding a cystein residue which can be readily used for disulphide, thioether, amine or acid bonds to one extremity of the polypeptide of the invention. A further approach consists of chemically coupling a biotin group, which then enables any substance bonded to streptavidin to be coupled. Coupling can also be effected using p-benzoquinone (FR-7537392 and U.S. Pat. No. 4,925,921, for example).
In general, any chemical, biochemical, enzymatic or genetic coupling method which is known in the literature can be used.
Further, a vector of the invention can comprise a polypeptide as described above to which a number of identical or different substances are coupled.
The examples below clearly demonstrate that the polypeptides of the invention have the ability not only to penetrate into cells, but also to transport substances of interest thereto. The examples demonstrate enzyme type protein transport. It should be understood that enzymes can be substituted by any other molecule of interest such as nucleic acids, peptides or drugs, under the same conditions.
The examples also demonstrate the capacity of the peptides of the invention to transfer nucleic acids into cells. For this particular application, coupling between the peptide and the nucleic acid is generally non-covalent coupling, based on ionic interactions, electrostatic interactions or Van der Waals forces. More particularly, when used to transfer nucleic acids into cells, a peptide of the invention advantageously comprises a region constituted by basic amino acids, for example lysine in type, enabling a complex (polyplex) to be formed with the negatively charged nucleic acids. Thus in one particular implementation, the invention concerns the use of a peptide as defined above, carrying a polylysine region, for transferring nucleic acids (i.e., plasmids, cosmids, linear fragments, genes, antigens, antisense, oligonucleotides, etc) into cells. In one particular aspect, the invention thus provides a peptide comprising a polylysine region and a region derived from a penetrating polyreactive antibody, and capable of penetrating into cells. More particularly, the polylysine region advantageously comprises 5 to 30 lysine residues, preferably 5 to 20, which are advantageously not interrupted by other residues. The region derived from the penetrating antibody can be defined as above. Such a polypeptide advantageously comprises less than 100 residues, as explained above.
Further, the fact that the polypeptides of the invention enable massive transport of proteins into cells has also prompted the Applicant to examine the possibility of using them as an intracellular antigen transport agent, endowing them with an adjuvant effect and leading to an increase in the immune response against these antigens. Thus mice received several injections with the streptavidin-peroxidase conjugate alone or complexed with a polypeptide of the invention. The results obtained show that the use of a polypeptide of the invention can increase by on average 4 to 8 times the titer of anti-streptavidin antibodies and anti-peroxidase antibodies.
The invention also concerns a method for transferring a substance into a cell in vitro, ex vivo or in vivo comprising:
coupling said substance to a polypeptide as defined above; and
bringing the cell into contact with the product of said coupling.
For in vitro or ex vivo use, contact can be effected by simple incubation of the cells with the coupling product (vector). For in vivo use, contact is generally effected by administering the coupling product (vector) to the organism under consideration. As indicated above, when coupling is covalent in type, the vector can comprise one or more molecules of interest. Further, for non-covalent coupling, for example for nucleic acids, the vector is generally formed by incubating the peptide and the nucleic acids in a medium enabling them to form a complex. The respective quantities of the partners are easily adjusted by the skilled person as a function of the nature of the peptide (length and charge), nucleic acid and cell type. By way of example, coupling can be carried out at peptide concentrations of 0.01 to 100 nmoles of peptide per xcexcg of nucleic acid, preferably 0.01 to 10 nmoles/xcexcg. Further, in the method of the invention, it may be advantageous to use, in addition, a stabilising agent or facilitator such as glycerol. Thus the results shown in the examples show that in the presence of glycerol, the transfection efficacy of nucleic acids can be improved by a factor of close to 40. Thus a particular implementation of the method of the invention comprises bringing cells into contact with the coupling product in the presence of a stabilising agent, in particular glycerol. Advantageously, transfection is carried out in vitro or ex vivo in the presence of glycerol, at concentrations or 0.1 to 2 M, for example. It should be understood that these concentrations can be adjusted by the skilled person.
In a still further aspect, the invention provides a cell, in particular a eukaryotic cell, containing a polypeptide or a vector as defined above. This cell is advantageously a mammalian cell, in particular an animal or human cell. In particular, it may be a cell of the hematopoietic system, such as a progenitor cell or a strain cell or a lymphocyte cell (T, B). It may also be a cell presenting the antigen such as macrophages or dendritic cells.
Further, the inventors have also shown that the polypeptides of the invention and the polyreactive antibodies are endowed with their own biological properties. The invention demonstrates that these polypeptides (peptide derivatives) or antibodies are capable of inducing a biological effect which is distinct from their ability to vectorise active substances. Unexpectedly, the present invention shows in particular that these antibodies and peptide derivatives are capable by themselves of exerting an antiviral activity on different cell populations and on different types of virus.
The present invention thus also concerns a novel approach to inhibiting viral replication and/or infection in cells. In particular, the present invention concerns the use of particular antibodies or antibody fragments as antiviral agents, in particular to inhibit viral replication and/or infection in cells. The invention also describes a novel method for treating cells to render them more resistant to viral replication and/or infection. The invention also concerns populations of cells treated by antibodies or polypeptides which are less sensitive to viral replication and/or infection. This property can be implemented in vitro, ex vivo or in vivo, optionally in combination with other agents, to reduce infection and development of a virus, in particular infection and/or replication of the human acquired immunodeficiency virus, polio virus, herpes virus or cytomegalovirus, for example.
Thus in a yet still further aspect the invention concerns the use of one or more polypeptides as defined above in preparing an antiviral composition. More particularly the invention concerns the use of one or more antibodies or antibody fragments in preparing an antiviral composition, characterized in that said antibodies or antibody fragments are polyreactive and are capable of binding to a nucleic acid, preferably DNA.
More particularly, an antiviral composition as defined in the invention consists of a composition which is capable of inhibiting infection of a target cell by a virus and/or replication of a virus in a target cell.
As indicated above, this aspect of the present invention stems from the demonstration of the unexpected biological properties of the polypeptides described above and, more generally, of certain polyreactive antibodies, i.e., susceptible of recognising a plurality of antigens, and more specifically having the capability to bind DNA.
In a more particular aspect, then, the invention concerns the use of polyreactive antibodies or antibody fragments, or anti-DNA and penetrating derivative polypeptides, as an antiviral agent.
As indicated above, the polyreactive antibodies or antibody fragments used are preferably capable of recognising at least one proteic antigen of cellular and/or viral origin. More particularly, in addition to DNA, these antibodies or antibody fragments recognise at least one viral antigen such as a viral protein envelope antigen. In one particular implementation, the invention concerns the use of polyreactive antibodies or antibody fragments which are capable of binding a protein or a peptide of the human acquired immunodeficiency virus (HIV). This property can be tested using any conventional immunological technique. Thus HIV proteins or peptides can be immobilised on any suitable support (plate, column, beads, etc.), and to incubate this support with the antibodies. The formation of an antigen-antibody complex can then be detected using any conventional technique (immunofluorescence, enzymatic reaction, etc.). In general, the avidity for HIV proteins or peptides of the anti-DNA antibodies used in the invention is of the order of 106 to 107 M.
Preferably, the antibodies used have the capability of binding HIV Tat and/or rev proteins, more preferably the Tat protein or a peptide of that protein. Particular peptides which can be cited are peptides comprising residues 22-37 and 46-60 of the HIV Tat protein.
In this antiviral application, the antibodies used can be polyclonal or monoclonal antibodies. Polyclonal antibodies can be isolated directly from the serum of subjects (immunised or not immunised, healthy or diseased) by removing blood and isolating antibodies in the presence of immunoadsorbents (for example protein A coupled to a sepharose type support). Polyclonal antibodies which can be used in the invention can in particular be obtained from healthy or diseased animal serum, in particular from rodents, more particularly from mice. They may in particular be autoimmune lupic mice which have high levels of natural antibodies recognising various antigens. Polyclonal antibodies can also be obtained from human serum, for example from patients with disseminated lupus erythematosus, which are also known to present a high natural level of polyreactive antibodies. Concerning monoclonal antibodies, they can be prepared using conventional immunological techniques, by removing splenocytes from immunised or non immunised animals, healty or pathological, fusion with myeloma cells, then clonal dilution and selection of hybridomas producing antibodies. These techniques have been widely documented, in particular by S. L. Morriso and V. T. Oi, in xe2x80x9cAdvances in Immunology (1989), 44: 65-92; J. G. R. Hurrell, xe2x80x9d Monoclonal Hybridoma Antibiotics: Techniques and Applicationsxe2x80x9d, CRC Press 1982. These techniques have also been illustrated in the examples. Polyreactive monoclonal antibodies can also be artificially synthesised or humanised from animal monoclonal antibodies.
The antibodies used can be immunoglobulins of different types, in particular IgG or IgM. It should be understood that other types of antibodies can also be used (IgE, IgA, etc.) provided that they have the required properties.
Further, as indicated above, the use of not only intact antibodies but any polypeptide of the invention is also possible, also any fragment of these antibodies which retains the required properties. These fragments can also be (Fabxe2x80x2)2, (Fabxe2x80x2) or ScFv fragments. These fragments can result from enzymatic antibody digestion, or can be obtained by recombination or by synthesis. The preparation of such fragments (for example by enzymatic treatments) has been widely documented in the literature and can thus be carried out by the skilled person using simple routine operations starting from antibody preparations.
As indicated above, this aspect of the present invention stems in part from the discovery of the antiviral properties of such polypeptides, antibodies and fragments, in particular their ability to inhibit viral replication in a target cell or infection of a target cell by a virus. The term xe2x80x9cinfectionxe2x80x9d means penetration of the virus or viral genome into the target cell, and the term xe2x80x9creplicationxe2x80x9d essentially means replication of the viral genome in said target cell.
The present invention can be employed to inhibit the cycle of different viruses, more particularly a RNA virus (retrovirus) or a DNA virus. Further, it may be a virus with tropism for man or for different animals, in particular mammals (dogs, cats, rabbits, cattle, etc.) More preferably, the present invention can inhibit a virus such as the human immunodeficiency virus (HIV), polio virus, herpes virus or cytomegalovirus (CMV).
A particular implementation of the invention comprises the use of one or more polyreactive antibodies or fragments of such antibody for preparing a composition intended to inhibit infection of a target cell by HIV and/or replication of HIV in a target cell, characterized in that said antibodies or antibody fragments have the ability to bind DNA. More preferably, the antibodies used also have the ability to bind a protein or a peptide of the human acquired immunodeficiency virus. More particularly, the invention can be used to inhibit infection and/or replication of different strains of HIV, in particular HIV-1 and HIV-2.
More particularly, the invention concerns the use of one or more polypeptides as described above to prepare a composition intended to inhibit viral replication and/or infection.
Within the context of the invention, the term xe2x80x9ctarget cellxe2x80x9d means any cell which is naturally susceptible of being infected by a virus, preferably susceptible of enabling replication of the virus. In the case of HIV, the target cells are constituted by cells of the immune system, in particular lymphocytic cells. More specific examples of HIV target cells are T lymphocytes, in particular auxiliary T lymphocytes (CD4+). Other HIV target cells for the invention are more generally constituted by peripheral mononuclear cells, in particular human (PBMC). Regarding the polio virus, an example of target cells are epithelial cells. In general, the present invention can be employed to interfere with the development of a virus in any type of target cell for the antibodies used.
Advantageously, a polypeptide of the invention is derived from recombinant ScFv fragments, capable of reacting with DNA or with other anionic or cationic macromolecules, in particular heparin and heparin sulphate, and obtained from lymphocytes originating from normal patients or patients with different diseases in particular disseminated lupus erythrematosus.
The mechanism(s) for the action of the compounds of the invention still have to be elucidated. In this respect, a series of recent results, in particular for the herpes virus, in particular the human herpes virus for example the type 1 herpes simplex virus or cytomegalovirus (CMV) appears to indicate that the peptides of the invention affix to cellular receptors used by the virus themselves to penetrate into the host cells. Fixing of peptides on these receptors is followed by internalisation of the peptide-receptor complexes, which results in a reduction in the number of cellular receptors remaining available to fix the virus. This reduction sometimes appears to be especially significant as in the case of CMV. However, other mechanisms, in particular on the level of the nucleus, could also be involved in the unexpected properties of these antibodies and derivatives.
Advantageously, the antiviral activity of the polypeptides means a significant reduction in replication or infection. Advantageously, the inhibition produced by the polypeptides or antibodies of the invention corresponds to a reduction by a factor of at least 1.5 with respect to the level of infection and/or replication in the same cells or cell populations in the absence of treatment. More preferably, inhibition corresponds to a reduction by a factor of at least 4. This inhibition can be quantified, for example by measuring the viral plaques, the levels of viral antigens present in the cells, cell viability, etc . . . In a particular implementation, the inhibition efficacy is evaluated by measuring the levels of viral antigens such as p24 and/or gp120 antigens, for HIV.
In a particular implementation, the invention concerns the use of antibodies or polypeptides to induce an inhibition of a factor of at least 2 in viral replication in the target cells.
In a first implementation, the invention comprises using a single type of polypeptide or antibody or antibody fragment as defined above. As illustrated in the examples, by using a single compound it is in fact possible to inhibit HIV-1 replication in target cells by a factor of more than 10, in particular of the order of 100. The use of a single compound can also produce an inhibition by a factor of more than 2 in the replication of type-1 polio virus in target cells.
Further, a pronounced anti-herpetic reaction has been observed for certain antibodies and peptides of the invention. It is important to indicate that this action is also observed with HSV-1 thymidine-kinasexe2x88x92 (TKxe2x88x92) on which acyclovir has no effect.
Further, a pronounced anti-CMV action has also been observed, in particular with the K19-pF4-1 polypeptide. In a series of experiments, it has been demonstrated that K19-pF4.1 inhibited the infection of cells by CMV by almost 100%. It is important to note that even the synthesis of early antigens was completely inhibited (detection both by immunofluorescence and radiolabelling, followed by immunoprecipitation).
In a further implementation, the invention comprises the use of a plurality of polypeptides, antibodies and/or antibody fragments as defined above. As illustrated in the examples, certain of these compounds, in combination, can exert a synergistic inhibition effect on viral replication in target cells. Thus, unexpectedly, certain antibodies, alone, have a moderate inhibiting activity but in combination induce an inhibition of HIV-1 replication in target cells by a factor of more than 10, in particular of the order of 100.
In a further implementation, the invention also comprises the use of one or more polypeptides, antibodies and/or antibody fragments as defined above in combination with one or more antiviral agents. Examples of such antiviral agents are AZT, DDI and antiproteases. In this regard, the present application also concerns a product comprising:
one or more polypeptides, antibodies and/or antibody fragments as defined above; and
an antiviral agent; for simultaneous, separate or intermittent use.
The present invention can be used to inhibit viral replication and/or infection in vitro, ex vivo or in vivo.
For in vitro or ex vivo use, the target cells, or a cell composition comprising target cells, are generally incubated in the presence of compounds as defined above. The doses of the polypeptides, antibodies or antibody fragments used are generally in the range about 1 to 500 xcexcg per 106 cells, preferably about 1 to 100 xcexcg/106 cells. These doses can of course be adapted by the skilled person without difficulty. Incubation is carried out in any suitable cell culture medium, and under the normal temperature conditions (for example between ambient temperature and about 37xc2x0 C.). The media used are any mammal cell culture media known to the skilled person, such as RPMI, DMEM, MEM, etc . . . Incubation can be carried out using any suitable apparatus such as a dish, flask, ampoule, pouch, tube, syringe, etc., preferably under sterile conditions. Advantageously, incubation is carried out for a period in the range about 1 hour to about 5 days, depending on the use and aim. As an example, cells can be incubated for a period in the range from about 1 hour to about 12 hours. The incubated cells can then be administered to a subject (autologous), and the subject can also receive one or more administrations of antibodies or antibody fragments.
For in vivo use, the compounds can be administered by different routes, such as systemic, intramuscular, or sub-cutaneously, for example. Preferred routes are systemic (in particular i.v.) and sub-cutaneous. The doses used can also be adapted by the skilled person as a function of the stage of the subject, the desired aim and the number and/or frequency of administrations.
Preferably, the invention is employed to inhibit viral infection or replication in target cells ex vivo. To this end, target cells are removed from a subject (PBMC, for example), incubated ex vivo with compounds as defined above (for example for 1 to 6 hours, at 37xc2x0 C., in a sterile pouch), then re-administered to the subject. The subject can also receive one or more administrations of the compounds, optionally in combination with one or more other antiviral agents. The compounds or compositions as described above are particularly suitable for preventive use, i.e., to inhibit viral replication or infection in healthy or seropositive subjects but who have not developed the symptoms of the disease. The invention can also be used as a maintenance treatment, used alone or in combination with other antiviral agents, as explained above.
The invention thus also concerns a method for improving the efficacy of antiviral agents comprising the combined use of polypeptide, antibody or antibody fragments as defined above.
The invention also concerns a method for modifying a cell with the aim of reducing infection of this cell by a virus and/or replication of a virus in this cell (i.e., to improve viral resistance of this cell), comprising bringing said cell into contact with one or more polypeptides, antibodies or antibody fragments as defined above. The invention also concerns any population of cells incubated in the presence of polypeptides, antibodies or antibody fragments as defined above. More particularly, such populations can be PBMC cells or other cells of the immune system, optionally packaged in a sterile container. Preferably, such a cellular composition generally comprises 105 to 108 cells. These cellular compositions can be used to study the viral cycle, to search for inhibiting compositions or associations of inhibiting compounds, or optionally to reduce the risks and effects of a viral infection in vivo after administration.
The invention also concerns a pharmaceutical composition comprising, in association with a physiologically acceptable vehicle, a vector as defined above in which the substance is an active principle of a drug.
The invention still further concerns a vaccine comprising, in association with a physiologically acceptable vehicle, a vector as defined above in which the substance is an antigen.
The present invention will now be described in more detail using the following non limiting examples which are provided by way of illustration.